Fluorescence-mediated molecular tomography

ABSTRACT

This invention relates to a fluorescence-mediated molecular tomographic imaging system, designed to detect near-infrared fluorescence activation in deep tissues. The system can use targeted fluorescent molecular probes or highly sensitive activatable fluorescence molecular probes. Such probes add molecular specificity and yield high fluorescence contrast, to allow early detection and molecular target assessment of diseased tissue, such as cancers, in vivo. The new tomographic imaging system enables three-dimensional localization in deep tissues and quantitation of molecular probes.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.12/126,745, filed on May 23, 2008, which is a continuation of U.S.patent application Ser. No. 10/443,463, filed on May 22, 2003, which isa continuation of PCTUS0144764, filed on Nov. 27, 2001, which is acontinuation in part and claims priority from U.S. patent applicationSer. No. 09/723,033, filed on Nov. 27, 2000, the contents of both ofwhich are incorporated herein by reference in their entireties.

TECHNICAL FIELD

This invention relates to extracting quantitative, three-dimensionalmolecular information from living mammals and patients usingfluorochromes and new optical tomographic imaging methods.

BACKGROUND

Molecular imaging can be broadly defined as the characterization andmeasurement of biological processes at the cellular and molecular levelin mammals and human patients. In contradistinction to “classical”diagnostic imaging, for example, magnetic resonance (MR), computedtomography (CT), and ultrasound (US) imaging, molecular imaging analysesmolecular abnormalities that are the basis of disease, rather thanimaging the end-effects of these molecular alterations. Specific imagingof molecular targets allows earlier detection and characterization ofdisease, as well as earlier and direct molecular assessment of treatmentefficacy. Molecular imaging can theoretically be performed withdifferent imaging technologies, up to now preferably with nuclearimaging technologies, e.g., PET and SPECT imaging) which have highsensitivity of probe detection. The IV administered imaging probestypically recognize a given target. Alternatively, some probesdetectable by MR imaging have been developed (Moats et al., AngewandteChemie Int. Ed., 36:726-731, 1997; Weissleder et al., Nat. Med.,6:351-5, 2000), although their detection threshold is generally in themicromolar instead of the pico/femptomolar range of isotope probes.

An alternative method is to use fluorescent probes for targetrecognition. For example, enzyme activatable fluorochrome probes aredescribed in Weissleder et al., U.S. Pat. No. 6,083,486, and fluorescentmolecular beacons that become fluorescent after DNA hybridization aredescribed in Tyagi et al., Nat. Biotechnol., 16:49-53, 1998.Fluorescence activatable probes have been used in tissue culture andhistologic sections and detected using fluorescence microscopy. Whenadministered in vivo, fluorescence activatable probes have been detectedby surface-weighted reflectance imaging (Weissleder et al., Nat.Biotechnol., 17:375-8, 1999); Mahmood et al., Radiology, 213:866-70,1999. However, imaging in deep tissues (>5 mm from the surface), inabsorbing and scattering media such as mammalian tissues, andquantitating fluorescence (and in particular fluorescence activation)has not been described.

To image light interactions in deeper tissues, light in the nearinfrared (near-IR or NIR) instead of the visible spectrum is preferred.Imaging with near infrared (near-IR or NIR) light has been in thefrontier of research for resolving and quantifying tissue function.Light offers unique contrast mechanisms that can be based on absorption,e.g., probing of hemoglobin concentration or blood saturation, and/orfluorescence, e.g., probing for weak auto-fluorescence, or exogenouslyadministered fluorescent probes (Neri et al., Nat. Biotech.,15:1271-1275, 1997; Ballou et al., Cancer Immunol. Immunother.,41:257-63,1995; and Weissleder, 1999). In either application, NIRphotons undergo significant elastic scattering when traveling throughtissue. This results in light “diffusion” in tissue that hindersresolution and impairs the ability to produce diagnosticallyinterpretable images using simple “projection” approaches(transillumination), as in x-ray imaging.

During the last decade, mathematical modeling of light propagation intissue, combined with technological advancements in photon sources anddetection techniques has made possible the application of tomographicprinciples (Kak and Slaney, “Principles of Computerized TomographicImaging,” IEEE Press, New York, 1988, pp. 208-218); Arridge, InverseProblems, 15:R41-R93, 1999) for imaging with diffuse light. DiffuseOptical Tomography (DOT) uses multiple projections and deconvolves thescattering effect of tissue. DOT imaging has been used for quantitative,three-dimensional imaging of intrinsic absorption and scattering (see,e.g., Ntziachristos et al., Proc. Natl. Acad. Sci., USA, 97:2767-72,2000), and also Benaron et al., J. Cerebral Blood Flow Metabol.,20(3):469-77, 2000). These fundamental quantities can be used to derivetissue oxy- and deoxy-hemoglobin concentrations, blood oxygen saturation(Li et al., Appl. Opt., 35:3746-3758, 1996) or hematoma detection indiffuse media.

Although intrinsic-contrast for DOT imaging may be useful in certainsituations, e.g., for functional brain activation studies or hematomadetection, these applications do not allow the extraction of highlyspecific molecular information from living tissues. Fluorochromeconcentration has been measured by absorption measurements(Ntziachristos et al., 2000) or by fluorescence measurements in phantoms(Chang et al., IEEE Trans. Med. Imag., 16:68-77, 1997; Sevick-Muraca etal., Photochem. Photobiol., 66:55-64, 1997). However, previouslydescribed DOT systems and/or image algorithms have not been useful toobtain three-dimensional quantitation of fluorescence in deep tissues inliving mammals.

SUMMARY

The invention is based on the discovery that in vivo fluorochromesignals from specific targeted molecular probes, e.g., probes targetedfor specific enzyme activities or DNA sequences, can be localized inthree dimensions in deep tissues and be quantitated with highsensitivity using a specially designed imaging system for this purposeand relying on self-calibrated image reconstruction, and new algorithmsto extract molecular maps.

In general, the invention features a near-infrared,fluorescence-mediated molecular tomography (FMT) imaging system thatincludes a NIR light source to provide incident light; a multipointincident illumination array to direct light into an object, e.g., ananimal or human patient, from two or more separate excitation points;multiple optic fibers to transmit light from the light source to eachpoint in the multipoint incident illumination array; a multipointdetection array to collect light, e.g., fluorescent light, emitted fromthe object from two or more separate collection points; atwo-dimensional emitted light array to transmit light emitted from theobject to a detector; multiple optic fibers to transmit light from eachcollection point to a corresponding point on the two-dimensional emittedlight array; and a detector to detect and convert light emitted fromeach point of the two-dimensional emitted light array into a digitalsignal corresponding to the light emitted from the object.

In this system, the emitted light can be continuous wave (CW) light,time-resolved (TR) light, or both CW and TR light.

The system can further include a processor that processes the digitalsignal produced by the detector to provide an image on an output device.The output device can provide multiple images simultaneously. Theprocessor can be programmed to process the digital signal by i)generating a corrected fluorescence measurement by subtracting abackground signal and filter bleed-through signal from collectedfluorescence measurements; ii) generating a corrected intrinsic signalmeasurement by subtracting a background ambient light signal fromcollected intrinsic signal measurements; iii) generating aself-calibrated fluorescence measurement by dividing the correctedfluorescence measurement by the corrected intrinsic measurement; iv)generating a corrected background-medium diffuse signal by subtractingthe collected background ambient light signal from a collected diffusesignal; and v) generating a self-calibrated intrinsic measurement bydividing the corrected intrinsic signal measurement by the correctedbackground-medium diffuse signal.

In other embodiments, the processor can be programmed to process thedigital signal by i) generating a self-calibrated measurementM=M1−M3/M2−M4, wherein M1 is an emission wavelength fluorescence signal,M2 is an intrinsic signal, M3 is a background bleed-through signal, M4is a background ambient light signal; ii) generating a self-calibratedintrinsic measurement M′=log(M2−M4)/(M5−M4), wherein M5 is abackground-medium diffuse signal; iii) minimizing a functionF(U)=(M−P×U)2 to obtain a distribution and magnitude of U, wherein U isa vector of unknown concentration of a target in the object being imagedand P is a forward predictor of M calculated by solving a diffusionequation for an appropriate geometry and background medium influorescence mode; iv) minimizing a function F′(O)=(M′−P′×O)2 to obtaina distribution and magnitude of O; wherein O is a vector of unknownconcentration of a fluorophore in the object, and P′ is a forwardpredictor of M′ calculated by solving a diffusion equation for theappropriate geometry and background medium in absorption/scatteringmode; v) calculating an activation ratio AR=U/O; and vi) generating animage corresponding to AR.

The imaging system can include more than 100 optic fibers to transmitlight into the patient and/or from each collection point of thedetection array, and the detector array can include at least 100collection points.

In this imaging system, the two-dimensional emitted light array cantransmit to the detector a two-dimensional pattern of multiple points oflight corresponding to light emitted from the patient inthree-dimensions, wherein the pattern varies over time at a ratecorresponding to switching of illumination from one to another of thetwo or more excitation points. In addition, the two or more excitationpoints are illuminated by the light source one at a time. In certainembodiments the NIR light directed into the object can be at awavelength of from 550 to 950, e.g., 670 or 750 to 850, nanometers, andthe detector can be a charge-coupled device (CCD) camera or include aphotomultiplier tube.

The system can also include the NIR fluorescent (NIRF) molecular probesthemselves. The probes can be activatable molecular probes.

The invention also features a method for displaying an optical molecularmap corresponding to a ratio of a concentration of a molecular probecomprising a fluorophore administered to a patient to a concentration ofan activated fluorophore corresponding to a specific target in thepatient by: i) providing a first data set of fluorophore concentrationbased on intrinsic absorption; ii) providing a second data set ofactivated fluorophore concentration based on fluorescence; iii) dividingthe first data set by the second data set on a point-by-point basis toprovide a third data set; and iv) processing the third data set toprovide an optical molecular map corresponding to a ratio of aconcentration of a molecular probe comprising a fluorophore to aconcentration of an activated fluorophore corresponding to a specifictarget in the patient.

In another aspect, the invention features a method of obtaining athree-dimensional, quantitative, molecular tomographic image of a targetregion within a patient, by administering a near-infrared fluorescent(NIRF) molecular probe to the patient, wherein the molecular probeselectively accumulates within a target region in the patient; directingnear-infrared light from multiple points into the patient; detectingfluorescent light emitted from the patient; and processing the detectedlight to provide a three-dimensional image that corresponds to thethree-dimensional target region within the patient and to the quantityof molecular probe accumulated in the target region.

In this method, the three-dimensional image can be visualized on atwo-dimensional output device. The processing can include digitizing thefluorescent signal emitted from the patient, self-calibrating thedigital signal by combining fluorescent and intrinsic signalmeasurements from the patient and background medium, and reconstructinga three-dimensional, quantitative image. In certain embodiments, theprocessing includes i) generating a corrected fluorescence measurementby subtracting a background signal and filter bleed-through signal fromcollected fluorescence measurements; ii) generating a correctedintrinsic signal measurement by subtracting a background ambient lightsignal from collected intrinsic signal measurements; iii) generating aself-calibrated fluorescence measurement by dividing the correctedfluorescence measurement by the corrected intrinsic measurement; iv)generating a corrected background-medium diffuse signal by subtractingthe collected background ambient light signal from a collected diffusesignal; and v) generating a self-calibrated intrinsic measurement bydividing the corrected intrinsic signal measurement by the correctedbackground-medium diffuse signal.

The processing can also include i) generating a self-calibratedmeasurement M=M1−M3/M2−M4, wherein M1 is an emission wavelengthfluorescence signal, M2 is an intrinsic signal, M3 is a backgroundbleed-through signal, M4 is a background ambient light signal; ii)generating a self-calibrated measurement M′=log(M2−M4)/(M5−M4), whereinM5 is a background-medium diffuse signal; iii) minimizing a functionF(U)=(M−P×U)2 to obtain a distribution and magnitude of U, wherein U isa vector of unknown concentration of a target in the object being imagedand P is a forward predictor of M calculated by solving a diffusionequation for an appropriate geometry and background medium influorescence mode; iv) minimizing a function F′(O)=(M′−P′×O)2 to obtaina distribution and magnitude of O; wherein O is a vector of unknownconcentration of a fluorophore in the object, and P′ is a forwardpredictor of M′ calculated by solving a diffusion equation for theappropriate geometry and background medium in absorption/scatteringmode; v) calculating an activation ratio AR=U/O; and vi) generating animage corresponding to AR.

In these methods, the molecular probes can be administered systemicallyor locally by injecting a molecular probe, e.g., an activatable probe.The molecular probe can be locally injected into the target region orinto a non-target region, for example, by intraperitoneal administrationwith systemic absorption and administration by an implanted slow-releasecompound or device such as a pump.

In certain embodiments of the new methods, the NIR light can be directedinto the patient from at least 32 separate points of light arranged in afixed three-dimensional geometry, or with a multipoint incidentillumination array comprising a belt having at least 12 points of light.In addition, the spatial localizations of the multipoint incidentillumination array and the multipoint detector array can be determinedby image co-registration. In other embodiments, photon pulses aredirected into the patient and the arrival of photons emitted from thepatient is time-resolved using a separate array of photon detectors.

The emitted fluorescent light in these methods can be continuous wave(CW) light, time-resolved (TR) light, or both CW and TR light. Inaddition, the methods can be performed dynamically as function of time,and the image can be co-registered with an image obtained by magneticresonance or computed tomography imaging. The multipoint incidentillumination array (or detector array) can include a fiducial, andwherein the fiducial is used to determine the spatial localization ofthe array on the object.

The invention also features a method of detecting a cellular abnormalityin a patient by using molecular probes targeted to a particular cellularabnormality, e.g., associated with a disease such as cancer, acardiovascular disease, AIDS, a neurodegenerative disease, aninflammatory disease, or an immunologic disease. The invention alsofeatures a method of assessing the effect of a compound on a specifiedmolecular target by using a molecular probe that is activated by themolecular target, wherein the probe is contacted to the target, thetarget is imaged prior to and after contact with the molecular probe,and the corresponding images are compared, wherein a change in themolecular target indicates the compound is effective. For example, thespecified molecular target can be a protease, and the compound can be aprotease inhibitor.

A molecular probe is a probe that is targeted to a molecular structure,such as a cell-surface receptor or antigen, an enzyme within a cell, ora specific nucleic acid, e.g., DNA, to which the probe hybridizes. Afluorophore is an agent that fluoresces. A fluorochrome is an agent thatfluoresces (e.g., a fluorophore) and has a color.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

The new methods and systems provide various advantages. For example, thenew methods and systems provide for the first time the ability to detectfluorescence activation, e.g., by enzyme activation, in deep tissue andto provide localization and quantitation in three dimensions. Inaddition, the new methods provide non-invasive, molecular imaging toprovide information at subcellular levels.

The impact of the new molecular imaging techniques is potentiallyenormous. First, the new methods and systems can provide insight intospecific molecular abnormalities that form the basis of many diseases,e.g. up-regulated proteases, other enzymes, cell surface receptors,cyclins, cytokines or growth factors in cancer. Second, the new methodscan be used to assess efficacy of novel targeted therapies at amolecular level, long before phenotypic changes occur. This, in turn, isexpected to have an impact in drug development, drug testing, andchoosing appropriate therapies and therapy changes in a given patient.Third, the new molecular imaging/quantitation methods and systemspotentially enable one to study the genesis of diseases in the intactmicroenvironment of living systems. Fourth, the new methods offluorescence-mediated molecular tomographic imaging are useful fortesting novel drug delivery strategies. Fifth, the imaging methods allowone to gain three-dimensional information that is much faster to obtainthan is currently possible with time consuming and labor intensiveconventional, basic science techniques.

The new imaging systems and methods will have broad applications in awide variety of novel biologic, immunologic, and molecular therapiesdesigned to promote the control and eradication of numerous differentdiseases including cancer, cardiovascular, neurodegenerative,inflammatory, infectious, and other diseases. Furthermore, the describeddetection systems and methods will have broad applications for seamlessdisease detection and treatment in combined settings.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are examples of auto-quenched, activatable,near-infrared (NIR) fluorescent probes particularly suited for use inthe new methods.

FIG. 2A is a schematic of a new three-dimensional, fluorescencetomography apparatus.

FIG. 2B is a schematic diagram of a positioning device used inconjunction with the apparatus of FIG. 2A to hold an animal in properposition for imaging.

FIG. 2C is a picture of an optical imaging chamber of the system of FIG.2A. The imaging chamber positions the source and detector fibers.

FIGS. 2D and 2E are alternative embodiments of fiber-coupling systemsthat can be used in the new fluorescence tomography apparatus.

FIGS. 3A to 3F are a series of schematic diagrams of alternativeembodiments of multipoint incident light arrays including circulararrays (as also shown in FIG. 2A), planar arrays, curved arrays, moldedarrays, belt arrays, and catheter arrays. All of these embodiments canbe used with the system shown in FIG. 2A.

FIG. 4A is a schematic of a time-resolved, three-dimensionalfluorescence-mediated molecular tomography (FMT) system that can be usedin conjunction with the system of FIG. 2A.

FIGS. 4B-4D are a series of photos of a positional insert used in theimaging chamber of the system of FIG. 2A (as shown in FIG. 4B), in amagnetic resonance imaging MRI coil (FIG. 4C), and holding a mouse in anMRI coil (FIG. 4D).

FIG. 5 is flow chart of the steps used to process analog fluorescent andintrinsic (absorption) signal data in three dimensions to provide (i) avector U of concentrations of activated fluorescent probes within agiven volume, (ii) a vector D of concentrations of non-activated andactivated probes, and (iii) a vector AR which is the ratio of activatedover total NIRF probe.

FIGS. 6A-6C are a series of images representing an absorption map (6A),a fluorescence map (6B), and a molecular map showing the absorptionratio (AR).

FIGS. 7A and 7B are schematic diagrams illustrating absorption imagingat high resolution. FIG. 7A shows the phantom setup, and FIG. 7Billustrates the reconstructed image.

FIGS. 8A and 8B are schematic diagrams of the experimental setup toimage enzyme activity in three dimensions in a tissue-like medium usinga circular multipoint incident light array in cross-section (8A) and inthree dimensions (8B).

FIGS. 8C-8F are still images of a time-lapse video made of the enzymeactivity observed in the experimental setup shown in FIG. 8A at 20 (8C),50 (8D), 115(8E), and 200 (8F) minutes, respectively.

FIGS. 9A-9C are a series of images from a live mouse imaged at across-section through the region of an implanted human tumor. FIG. 9A isa T2-weighted MR image. FIG. 9B is a NIR fluorescence-mediated moleculartomography (FMT) image of the tumor obtained 24 hours after intravenousinjection of an activatable cathepsin B-reporting NIR imaging probe.FIG. 9C is a fused image that demonstrates the good co-registration ofthe tumor as it is seen on the T2-weighted MR image and on theNIRF-activated FMT image.

FIGS. 10A and 10B are a pair of images, MR and FMT, respectively, from alive mouse imaged at a cross-section at the level of the heart.

FIGS. 11A and 11B are a pair of images, MR and FMT, respectively, from alive mouse imaged at a cross-section at the level of the kidney.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

This invention relates to extracting quantitative molecular informationfrom living mammals and patients using fluorochromes, e.g., activatablefluorochromes, and a novel optical tomographic imaging method. Thisfluorescence-mediated molecular tomographic (FMT) imaging system isspecifically designed to detect NIR fluorescence (NIRF) activation indeep tissues with high sensitivity, quantitatively and over time. Thesystem can use activatable NIRF molecular probes that are quenched anddo not fluoresce until activated or highly sensitive targeted NIRFmolecular probes. The activatable molecular fluorochrome probes addmolecular specificity and yield high fluorescence contrast, to allowearly detection and molecular target assessment of cancers and otherdiseased tissue in vivo. The systems include various components forobtaining the image data and one or more processors that include newalgorithms to process the data to provide the high levels of informationand resolution.

The FMT imaging methods and systems enable extraction of molecularinformation from diseased tissue. Thus, the systems and methods can beused to detect many molecular aberrations, as they occur in cancer,cardiovascular disease, inflammation, immunological diseases, arthritis,cutaneous and ophthalmic diseases, and others.

After reviewing the suitable probes and the general methodology ofoptical imaging, we will describe the new imaging systems and theprocessing required to obtain useful three-dimensional, quantitativeinformation.

Activatable NIR Fluorescent Probes

A fundamental paradigm shift in injectable contrast agents has recentlybeen introduced by synthesizing probes that are become brightlyfluorescent following conversion by specific enzymes (Weissleder et al.,Nat. Biotechnol., 17:375-378, 1999) or become fluorescent by DNAhybridization (Tyagi et al., Nat. Biotechnol., 14:303-308, 1996). Intheir native state the probes are quenched either by a small moleculequencher (e.g., DABCYL (a non-fluorescent chromophore that serves as auniversal quencher for any fluorophore in a molecular beacon:4-(4-dimethylaminophenylazo)-benzoic acid) or QSY-7) or by multiplefluorochromes (e.g., through energy resonance transfer. FIGS. 1A and 1Bshow schematics of two probes designed to target a specific enzyme (1A)and a specific DNA sequence (1B). When the fluorochrome is released orspatially separated from its quencher, fluorescence can increase up to1000 fold. Because the spatial rearrangement of the quenchedfluorochromes occurs only after specific interactions, these probes canbe used to extract molecular information from living organism. Theseactivatable probes have four major advantages over other methods whensingle fluorochromes are attached to affinity molecules: (1) a singleenzyme can cleave multiple fluorochromes, thus resulting in one form ofsignal amplification, (2) reduction of background “noise” by severalorders of magnitude is possible, (3) very specific enzyme activities canpotentially be interrogated, and (4) multiple probes can be arranged ondelivery systems to simultaneously probe for a spectrum of enzymes.

A panel of highly specific enzyme sensitive molecular probes have beensynthesized that target matrix metalloproteinase-2 (MMP-2), cathepsinB/H, cathepsin D, cathepsin K, PSA, and caspase-3), and which arecapable of fluorescence activation at 600-900 nm. These probes aredescribed in detail in Weissleder et al., U.S. Pat. No. 6,083,486;Weissleder et al., Nat. Biotechnol., 17:375 (1999); Tung et al., CancerResearch, 60:4953-8, 2000; and Tung et al., Bioconj. Chem., 10:892-896,1999). The activatable sensitive probes typically consist of threebuilding blocks: (1) fluorochromes, (2) target substrate, and (3) adelivery vehicle.

Reporter Fluorochromes:

Hundreds of optical probes have been developed for microscopy andphotodynamic therapy. Of these, fluorescent probes (i.e., excitation atshorter wavelength and emission at longer wavelength) are ideally suitedfor studying biological phenomena, as has been done extensively influorescence microscopy. If fluorescent probes are to be used in livingsystems, the choice is generally limited to the near infrared spectrum(600-1000 nm) to maximize tissue penetration by minimizing absorption byphysiologically abundant absorbers such as hemoglobin (<550 nm) or water(>1200 nm). Ideally the fluorochromes are designed to emit at 800±50 nm.A variety of NIRF molecules have been described and/or are commerciallyavailable, including: Cy5.5 (Amersham, Arlington Heights, Ill.); NIR-1(Dojindo, Kumamoto, Japan); IRD382 (LI-COR, Lincoln, Nebr.); La JollaBlue (Diatron, Miami, Fla.); ICG (Akorn, Lincolnshire, Ill.); and ICGderivatives (Serb Labs, Paris, France). NIRF probes for in vivo useideally should have the following properties: (1) narrow emissionbandwidths, (2) high fluorescence efficiency (quantum yield), (3)biocompatibility, and (4) spectrally separated absorption andexcitation.

Target Substrates:

The release and or availability of individual fluorochromes isdetermined by interaction of a target substrate with its target. Atarget substrate can, for example, be a peptide sequence that is cleavedby enzymes (see Table 1 below), a phosphate group which is transferredby certain kinases, or a hybridizing DNA sequence recognizing a specificcomplementary DNA motif (see FIG. 1B).

TABLE 1 Examples of Peptide Substrates (dots indicate the cleavage site)Protease target Peptide sequence Cathepsin D GPIC(Et)F•FRLG Cathepsin BGRR•G Matrix metalloproteinase 2 GPLG•VRG Caspase 3 DEVD•G Prostatespecific antigen HSSKLQ•G

Delivery Vehicle:

For a quenched probe to reach its intended target, it has to evade rapidclearance/elimination and overcome several structural barriers todelivery. These barriers include: (1) extravasation from vessels, (2)diffusion through tissue, and (3) cell membrane translocation in thecase of intracellular enzymes (not required for secreted enzymes). Thesebarriers to delivery are fairly well investigated, and delivery vehiclescan be selected using standard techniques and information. Suitablevehicles to deliver fluorochromes and substrates to a target, e.g., atumor, in the body can be selected from a group of polymers, includingprotected graft co-polymers (Marecos et al., Bioconjug. Chem.,9:184-191, 1998) containing polyethylene glycol (PEG), polaxamers,and/or carbohydrates. Additional delivery vehicles include dendrimers,proteins, carbohydrates, lipid spheres (e.g., emulsions, liposomes, andlipid self-assemblies), nanoparticles, and other materials commonly usedfor parenteral drug delivery.

Specific probes based on the above design for use in the new methods canbe prepared as described in detail in Weissleder et al., U.S. Pat. No.6,083,486; Weissleder et al., Nat. Biotechnol., 17:375-8, 1999; and Tunget al., Bioconj. Chem., 10:892-896, 1999.

One specific example of enzyme activatable probes for use in the newmethods can be prepared as follows (see, Weissleder et al., U.S. Pat.No. 6,083,486; Weissleder et al., Nat. Biotechnol., 17:375-8, 1999). Aprotected graft copolymer (PGC) consisting of a poly-L-lysine (PL)backbone and methoxy poly-e-ethylene glycol (MPEG) side chains is firstsynthesized (Bogdanov et al., J. Drug Targeting, 4:321-330, 1997). Inone example, Cy5.5 (absorption=675 nm, emission=694 nm, Amersham,Arlington Heights, Ill.) can be directly attached to the poly-lysinebackbone, yielding an activatable probe that can be cleaved by cathepsinB/H and trypsin and has been used for the experiments described below.Briefly, an excess of monoactivated Cy5.5 was reacted with PGC at pH 8.0to yield the probe. The final products were separated from free dye bysize-exclusion chromatography. Trypsin and cathepsin B/H-like proteasesare capable of cleaving such probes as occasional free lysine residuesrepresent an enzyme substrate.

Alternatively, one can attach specific peptides conferring enzymespecificity directly to the PGC graft copolymer. For example, cathepsinD sensitive probes have been synthesized (Tung et al., Cancer Res., 60:4953-8, 2000 and Tung et al., Bioconj. Chem., 10:892-896, 1999).Briefly, PGC was reacted with large excess of iodoacetyl anhydride toconvert all amino groups on the polylysine backbone into iodol groups.The cathepsin D specific peptide, GIC(Et)FFKK(Fitc)C was attached to theiodinated PGC through a thiol specific reaction. Thereafter, Cy5.5 wasattached to the N-terminus and the free lysine side chains of thecathepsin D substrate peptide. The advantage of this design is twofold:(1) a high loading capacity (due to the fact that all lysines can bemodified), and (2) that the fluorochrome spacer is readily accessible toenzymes, thus resulting in improved release kinetics and signalrecovery.

Other NIR Fluorescent Probes

The probes described above are specifically designed to become activatedupon target interaction, e.g., target enzyme interaction. Alternativeprobes that can be used in the new detection methods include (1) probesthat become deactivated (quenched) after target interaction, (2) probesthat change their quantum yield upon target interaction, (3) probes thatchange their fluorescence lifetime after target interaction, (4) probesthat change their fluorescence spectrum after target interaction, (5)wavelength shifting beacons (Tyagi et al., Nat. Biotechnol.,18:1191-1196, 2000), (6) multicolor fluorescence probes (Tyagi et al.,Nat. Biotechnol., 16:49-53, 1998), or (7) probes that have high bindingaffinity to targets, i.e., that remain within a target region whilenon-specific probes are cleared from the body. Examples of the latterprobes include receptor-targeted NIR fluorochromes (Achilefu et al.,Invest. Radiol., 35:479-485, 2000) or antibody-targeted NIRfluorochromes (Ballou et al., Biotechnol. Prog., 13:649-658, 1997.Another group of suitable fluorescent probes are long lifetimelanthanide metal-ligand probes that will allow the use of gateddetection, and further increased sensitivity.

General Methodology

The new systems use a charge-coupled device (CCD) camera and lens systemto obtain “tomographic measurements” from the periphery of a multipointincident light array, such as a cylinder with numerous, spaced, lightemitters, for three-dimensional optical scans. Improvements in NIR imagequality are related to the number of sources and detectors used. Theadvantage of CCD technology is that increasing the detector density doesnot require additions in the detection hardware, just additional opticfibers to create a bigger array.

Fluorescence-Mediated Molecular Tomography (FMT)

The tomographic methodology described herein is an improvement of thegeneral category of tomography using diffracting sources (see, e.g., Kakand Slaney, “Principles of Computerized Tomographic Imaging,” IEEEPress, New York, 1988, pp. 208-218). The technique uses measurements oflight at multiple projections to obtain information of the opticalcontrast inside turbid media such as tissue. In brief, diffractiontomography segments the volume under investigation into a number ofdiscrete voxels, referred to as a “mesh.” The analysis is divided intotwo steps. The first step is the “forward problem,” in which a diffusionequation is used to describe the photon propagation into an assumedmedium, e.g., tissue, and is used to predict the field detected fromthis medium. The second step is the “inverse problem,” in which theoptical properties of each voxel of the assumed medium are updated tominimize the errors observed between the predicted and measured fields.There are several ways to calculate the forward problem (analytical andnumerical solutions of the diffusion equation) and inverse problem(direct inversion, χ²-based fits, and algebraic reconstructiontechniques). Here we use a numerical solution of the forward problem togenerate the prediction vectors for the fluorescence and intrinsicsignal measurements (See also FIG. 5). Inversion is based on the relaxedalgebraic reconstruction technique. Higher order solutions can beobtained if needed when a solution is fed back in the forward problem toproduce more accurate forward propagation models, and this process canbe repeated iteratively.

The new FMT imaging systems use one or more laser sources to detectspecific chromophores or fluorophores and the forward problem iscalculated for the specific wavelength(s) used. Laser diodes are used aslight sources since they produce adequate power, are within the FDAclass I and class II limits, and are stable, wavelength-specific andeconomical. Light is directed to and from tissue using fiber guides, asthis allows flexibility in the geometrical set-up used. For opticalcoupling the fibers have to be in contact with tissue. Alternatively,matching fluid is used to eliminate reflections due to air-silica-tissueindex of refraction mismatch.

Three different light source-detection technologies exist. Anycombination of them can be used for FMT applications as describedherein. The simplest is continuous wave (CW) imaging. This techniqueuses light of constant intensity and measures either (1) the signal dueto a distribution of excited fluorophores or (2) the attenuation oflight (due to tissue absorption and scattering) employing multiplesource-detector pairs. The technique is technically relatively simpleand usually offers the best signal-to-noise (SNR) characteristics.However, it is not best suited for imaging of intrinsic tissue contrastsince it usually introduces significant cross-talk between thecalculations and imaging of absorption and scattering coefficients. Onthe other hand, if the background optical properties are known, themethod is well-suited for imaging fluorophore concentration in thesteady-state. To produce activation information, a combination of thistechnologically simple approach with a technology richer in informationcontent can be used to obtain a both fluorescence and intrinsic contrastimages. A specific design is described below, in which the light sourceis switched from one light emitter to another on a multipoint array inseries, so that only one emitter is illuminated at a time.

A more elaborate approach is to use intensity modulated (IM) light at asingle or at multiple frequencies. With this method, modulated lightattenuation and phase shifts, relative to the incident light, can bemeasured for multiple source-detector pairs. Compared to a CWmeasurement, which yields intensity attenuation, the IM technique offerstwo pieces of information, i.e., intensity attenuation and phase shiftper source-detector pair. Amplitude and phase are usually uncorrelatedmeasurements and can more efficiently resolve the absorption andscattering coefficient of intrinsic contrast. In the fluorescence mode,the technique can image two sets of information, fluorophoreconcentration and fluorescence lifetime.

The third approach, the time-resolved (TR) technique, uses short pulsesof light injected into the tissue. The technique resolves thedistribution of times that the detected photons travel into the mediumfor multiple source-detector pairs. Time-resolved methods contain thehighest information content per source-detector pair, comparable only tothe IM method performed simultaneously at multiple frequencies. This canbe easily explained when one considers that the Fourier transform of thetime-resolved data yields information at multiple frequencies up to 1GHz, including the continuous wave components (f=0 MHz) used by theprevious two methods. Therefore, the time-resolved method offers a CWcomponent for direct comparison with the CW system, but also intensityattenuation and phase-shift measurements at multiple-frequencies (viathe Fourier transform) that can image intrinsic absorption andscattering, and also fluorophore concentration and fluorescencelifetime.

A cost-efficient embodiment of the invention is described in detailbelow (see FIGS. 2A-2C and FIG. 4). In this embodiment, the bulkinformation is collected using economical, massively parallel CWmeasurements (˜1000 channels) and highly specific information ofabsorption and scattering parameters are collected with a smaller arrayof time-domain source-detection channels (˜50-100 channels). Thetime-domain information is used in three ways. The first is toindependently quantify the average absorption and reduced scatteringcoefficient at the emission and excitation wavelength. The second is toimplement time-domain measurements of intrinsic signal into theintrinsic reconstruction scheme by Fourier transforming the time-domaindata, hence obtaining multiple-frequency readings. Since the tomographicproblem is written in the frequency domain (with CW measurements havingzero frequency) the addition of extra, higher frequency measurements isstraightforward (just adding additional lines in the weight matrixconstructed for the appropriate frequency, for both real and imaginarydecompositions). The third use of the time domain system is to implementtime-domain measurements of fluorescent signal into the fluorescentreconstruction scheme by Fourier transforming the time-domain data, andobtain information of the fluorescence lifetime of the NIRF probe.

Fluorescence-Mediated Molecular Tomographic (FMT) Imaging Systems

The new imaging systems include an apparatus with various componentsused to generate digital signal data from analog fluorescence emittedfrom a patient or animal body, and a processor programmed withalgorithms that can process the digital signal data into useful imagesthat provide diagnostic and prognostic information. The systems can alsoobtain measurements of the incident light after it propagates throughthe tissue and obtain information on the intrinsic contrast of the bodybeing imaged.

Apparatus

Diffraction tomography differs from simple projection imaging in that itrequires tissue transillumination at multiple projections. Therefore theconstruction of an appropriate light guiding apparatus is fundamental toobtain molecular tomographic images using NIR light. In one embodiment,the system features a multipoint incident illumination array and amultipoint detector array, both incorporated into a single cylinder, tobe placed around the animal or patient body. One such apparatus is shownin FIG. 2A and FIG. 4. The two instruments can operate sequentially.

System 10 includes a continuous wave (CW) laser source 12. The laser 12uses constant intensity light. Two wavelengths obtained from twodifferent lasers can be used for imaging the intrinsic contrast beforethe administration of the NIRF probe. For imaging the fluorochrome Cy5.5, one wavelength is set to 673 nm (excitation wavelength) and theother to 694 nm (emission wavelength). Imaging at both wavelengths isnecessary so that accurate forward models are created for the excitationfield from the source to the fluorophore and for the emission field fromthe fluorophore to the detector. The other combination of wavelengthswill target the fluorochrome ICG at 750 nm (excitation) and 800 nm(emission). The two wavelengths are time-shared since the measurementsare not very demanding in terms of time efficiency and are coupledthrough an optical attenuator 14 to a 1×32 optical switch 16 (e.g., anoptical switch from Dicon FiberOptics Irvine Calif.). The optical switch16 directs light from laser 12 to any one of multiple (sixteen in thisembodiment) source fibers 18. Alternatively, all fibers can beilluminated simultaneously, each at a different wavelength. The key isto be able to distinguish each point of illumination on the multipointincident illumination array 20.

In this embodiment, the multipoint incident illumination array 20 islocated within a resin cylinder 15 (also referred to herein as an“imaging chamber”), with several rings of multiple source fibers 18connected around the cylinder. In essence, cylinder 15 has numerousholes drilled into it in a series of “rings” at different levels of thecylinder and perpendicular to the central axis. The holes can be equallyspaced around the perimeter of the cylinder. The source fibers 18 passthrough the holes in cylinder 15 and end flush with the inner wall. Amultipoint detector array 21 is incorporated into the same cylinder 15,in the form of rings of detector fibers 22 interleaved (alternating)with the rings of the source fibers 18. Again, the cylinder has holesdrilled for each detector fiber. This provides three-dimensional volumecoverage within the cylinder. Detector fibers 22 form the detector array21 of cylinder 15, and, like the source fibers, end flush with the innerwall of the cylinder. In this first implementation, three rings oftwelve detection fibers each are interleaved with two rings of sixteensource fibers each, each ring at 3 mm from the next, thus covering atotal cylinder height of 1.2 cm.

Cylinder 15 (including multipoint incident illumination array 20 andmultipoint detector array 21) can be filled with a liquid opticalcontact medium (e.g., Intralipid® or an emulsion of TiO₂ particles andappropriate amounts of an absorbing fluorophore or fluorochrome thatsimulate the optical properties of the tissue examined), which serves asthe “coupling” fluid of diffuse photons from the surface of the animalbody to the detection fibers. The concentration of TiO₂ particles forthe matching fluid and the resin cylinder will be such as to inducescattering properties comparable with the average reduced scatteringcoefficient of mice.

Fluorescent light collected by multipoint detector array 21 is fedthrough detector fibers 22 to a two-dimensional emitted fluorescentlight array 24. The two-dimensional array 21 transmits the analogfluorescent light emitted from the body through a long-pass filter 25(depending on the fluorochrome used) and to a CCD camera 26. Thelong-pass filter 25 will be selected for the appropriate cut-offwavelength, similar as done for surface reflectance type on imagingsystems (Mahmood, Radiology, 213:866-870, 1999). To image intrinsiccontrast the filter is removed. The CCD camera 26 is mounted on abreadboard, and a lens 27, or a system of macro lenses, images thetwo-dimensional emitted fluorescent light array onto the CCD camera.

Optimum light attenuation will be set by the optical attenuator 14 sothat measurements will not saturate the CCD camera. For a typical 16-bitCCD camera the useful dynamic range is approximately three to fourorders of magnitude. This is also the dynamic range expected formeasurements of diffuse light in small animals with body diameters ofabout 2-3 cm. The dynamic range expected for human patients may differdepending on the target organ. For example, for human breast imaging, atan approximate diameter of 8 cm, the dynamic range required is about 6-8orders of magnitude. This dynamic range can be covered using CCDtechnology by rapidly acquiring multiple frames. With current CCDtechnology used at 10 frames per second, the dynamic range can be 6orders of magnitude in one second of acquisition. For brainmeasurements, higher dynamic range may be achieved with longeracquisition times or more time-efficiency by using programmableattenuators that selectively attenuate the higher signals with a knownlevel of attenuation.

Additionally a positional device 23 can be used for optimum placement ofthe animal in cylinder 15 as shown in FIG. 2B. The positional device inthis embodiment is simply a cylinder that fits snuggly within cylinder15. Three positional devices 23 (cylindrical inserts) have beenconstructed. The first insert is constructed of Lexan® (polycarbonate)or Plexiglas®, and the second is constructed of white Delrin®,Polypropylene, or Kel-F®. Both of these inserts have an outer diameterthat exactly fits the inner diameter of cylinder 20, 21, and are 1 mm inthickness. The third insert is constructed out of Mylar® film and Kel-F®film to produce an insert with a wall thickness of 0.1 mm diameter. Theadvantages of this design are that the animal is stabilized duringimaging and that positional accuracy with surface marks can beestablished for co-registration purposes.

A detailed view of cylinder 15 (the imaging chamber), including both themultipoint detector array 21 and the multipoint incident illuminationarray 20, is shown in FIG. 2C. The source fibers 18 and detector fibers22 are arranged so that measurements are obtained along the entirecylinder to allow for three-dimensional reconstructions. Source fibers18 are interleaved between the detector fibers.

FIGS. 2D and 2E illustrate two alternative fiber-coupling systems. FIG.2D shows the system used in FIG. 2A, in that a separate two-dimensionalemitted fluorescent light array 24 is used to collect the signals of alldetector fibers 22 in one plane, which is imaged by CCD camera 26through filter 25. FIG. 2E shows a simpler embodiment in which thedetector fibers 22 are directly connected to filter 25, i.e., filter 25serves as the two-dimensional array 24.

Other embodiments of the multipoint incident illumination array areshown in FIGS. 3A to 3F. FIG. 3A illustrates a top view of thecylindrical array described above. FIG. 3B shows a planar array used forreflectance and/or transmittance mode operation. In an alternativeembodiment, the array is a portion of a cylinder, e.g., in the form of acurve with a set radius as shown in FIG. 3C. On the other hand, FIG. 3Dshows a schematic of a molded array, in which the ends of the lightsource fibers are arranged on a rigid substrate that conforms to aspecific shape of a body, or are arranged on a substrate of bendable,elastic material, such as a plastic, rubber, or cloth that can securethe light emitting optic fibers, and that can be molded to conform to abody shape. FIG. 3E illustrates a belt-like, uneven array, in which theends of the source fibers are arranged in a flexible belt that can befastened around a patient or the limb of a patient as required. Theexact positions of the light emitting points within this array can bedetermined and corrected for by concomitant CT, US, or MR imaging. In analternative embodiment, the ends of the light source fibers are providedin a catheter-like device as shown in FIG. 3F.

In each of these embodiments, the ends of the detector fibers 22 can beinterleaved with the ends of the source fibers 18 as in the cylinder 15shown in FIG. 2A. Alternatively, the detector array can be separate anddistinct from the incident illumination array, as long as the ends ofthe detector fibers are spaced in a specified geometry with respect tothe ends of the source fibers. For example, in the catheter-like array,the preferred mode of use is with a separate detector array thatpositions the ends of the detector fibers on the outside of the bodywhile the incident light array is positioned inside the body, e.g., toimage the prostate gland, lungs, vasculature, or gastrointestinal tract.

The apparatus 10 of FIG. 2A is used with a processor 11, e.g., locatedin a PC, as described in further detail below. As shown in FIG. 4, sucha processor 11 generally includes an input/control device 60, a memory62, and an output device 64. The processor 11 can be an electroniccircuit comprising one or more components. The processor can beimplemented in digital circuitry, analog circuitry, or both, it can beimplemented in software, or may be an integrated state machine, or ahybrid thereof. Input/control device 60 can be a keyboard or otherconventional device, and the output device 64 can be a cathode ray tube(CRT), other video display, printer, or other image display system.Memory 62 can be electronic (e.g., solid state), magnetic, or optical.The memory can be stored on an optical disk (e.g., a CD), anelectromagnetic hard or floppy disk, or a combination thereof.

A highly efficient photon collection apparatus of FIG. 2A can be builtusing the same or similar components as discussed above, but with theexception that dedicated detector fibers 22 are directly coupled to theCCD (as shown in FIG. 2E), versus the lens system shown in FIGS. 2A and2D. Overall, this system design should provide at least 300% improvedphoton counting efficiency. Higher efficiency CCD chips will furtherimprove photon detection.

To achieve a higher image-resolution design the apparatus of FIG. 2A canaccommodate more source-detector pairs (for example 64×100) either by alens-imaging system (FIGS. 2A and 2D) or by direct coupling (as shown inFIG. 2E). The latter system could require a larger dimension chip CCDcamera to accommodate the larger detector set.

In use, baseline measurements can be obtained from the tissue at theexcitation wavelength and at the emission wavelength without using thefilter. Fluorescence measurements can be performed at the emissionwavelength after inserting the appropriate cut-off filter.

An add-on system that will significantly enhance the tomographicaccuracy is shown in FIG. 4A. This is a time-resolved FMT imaging system30. A 16×16 channel array is implemented together with CW measurementsto yield superior reconstructions. The CW and TR system can be usedindependently but a benefit is achieved when the measurements obtainedfrom both systems are combined in the same reconstruction scheme.

In general, system 30 includes a pulsed laser source 32, a wavelengthcoupler 34 and a wavelength splitter 36. Two sets of two pulsed laserdiodes (pulse width ˜70 picosecond, average power ˜150 μW) are employedat the same wavelengths as the proposed CW system of FIG. 2A. Thewavelengths are used time-multiplexed with 10 nm delays; they aredetected simultaneously by the 16-channel single photon countingtime-resolved system 44 (e.g., a SPC-600® from Pico-Quant, Berlin,Germany). The time-resolved system can share the same source fibers 18′as the CW system 18 (by connecting both CW and TR light sources to theoptical switch) or use separate, dedicated source fibers. Thetime-resolved (TR) detection fibers 22′ will be interlaced with the CWdetector fibers 22. The TR acquisition will be obtained at differenttimes than the CW acquisition to avoid cross-talk between the CW and TRsystems. The relatively small source-detector array 18′, 22′ of the TRsystem (which can also be incorporated into cylinder 15) is capable ofproducing useful diffuse images. However, the two main contributions ofthe TR data will be (1) their simultaneous implementation is theinversions of Eq. 1 to obtain multi-frequency information in additionwith the CW data offering a stand-alone CW-TR tomographer, but also (2)their use with the concurrent magnetic resonance (MR) information toobtain measurements of fluorescence concentration and lifetime from thetumor lesions as identified on the MR images.

The pulsed laser source 32 produces laser light that is coupled bywavelength coupler 34 and then split by splitter 36. The splitterdirects ˜99% of the laser light along path 39 a into the optical switch16 and 1% of the light along path 39 b into the detector module 40 viathe corresponding attenuators 38 a and 38 b. The light traveling alongpath 39 b from attenuator 38 b provides a “reference signal” that isused to monitor the system's temporal drifts and signal stability. The99% part of the laser light on path 39 a that is directed to the opticalswitch 16 is switched in the same manner as in the CW system to selectedsixteen (or more, if needed) CW source fibers 18. There is no need touse two different switches and source fibers. The same optical systemused for the CW system can be used to also direct the photon pulses ontothe tissue of investigation in the light chamber 15. Fibers 18 can be(but need not be) physically identical to fibers 18′ and the onlydifferentiation is made for ease of illustration to indicate theiroperation passing CW or TR signals. A 2-to-1 optical switch 16′, e.g.,provided within the Dicon switch 16, can select between the CW or TRsource. However, an independent TR detector fiber array (sixteen fibers)is required to direct the collected photons at the time resolveddetection system 44. Cylinder 15 is the same as in FIG. 2A. Fluorescentor intrinsic light emitted from the body is passed to the TR system asin FIG. 2A and to detector module 40. Fibers 22 indicate the detectorfibers of the CW system shown on FIG. 2A.

Detector module 40 includes photomutiplier tubes (PMT) 41 that detectphotons and convert single photons to electrical analog pulses. Theseanalog pulses pass to router 42, which directs the pulses via path 43 tothe SPC-600 board 44. Here the pulses are converted to digital valuesthat indicate the time of arrival (TOA) of each coming pulse relative tothe trigger pulse on path 52 coming from laser 32. Each pulse collectedgenerates in router 42 a digital address, which uniquely marks thedetection channel from which this photon was detected. This digitaladdress is directed to the computer memory 62 via digital cable 45 andis used to store the TOA in the appropriate memory bin allocated foreach individual channel. For the sixteen channels used in thisembodiment, there are sixteen separate digital addresses correspondingto sixteen separate memory bins. Within system 44, constant fractiondiscriminator CFD 50 rejects pulses that have a very small amplitude andare probably due to photo-electronic noise, the Time-to-AmplitudeConverter (TAC) converts the time of pulse arrival to an analogamplitude value, and the Multi-Channel Analyzer (MCA) converts thisanalog amplitude to a digital value at high speed. The output 47 ofsystem 44 is a digital value stored in the computer memory bin thatcorresponds to the address carried on cable 45.

Time-resolved measurements can be used independently to obtain averagebackground properties of the medium measured, an important inputparameter for absorption, scattering, and fluorescence reconstructions.The combination of TR and CW measurements will produce more accurateforward problems for the intrinsic contrast and fluorescencereconstructions. Furthermore, the simultaneous use of CW and TR datawill enhance the overall image quality and fidelity. Another alternativewould be to use the time-resolved data to produce low-resolution imagesof background intrinsic contrast and use this information to create moreaccurate forward problems for the CW reconstructions for each animal.

The CW and especially the TR information can further be combined with MRimaging data to produce accurate quantitative measures of fluorophoreconcentration and fluorescence life-time measurements. Time-resolvedmethods would significantly open the spectrum to differentiate thefluorescence decay of existing and novel fluorochromes distributed intissue. The cyanine fluorochromes that are described above typicallyhave decay times ranging from 1 to 20 ns. While this timescale is usefulfor many biophysical measurements, there are numerous instances wherelonger decay times are desirable. For instance, one may wish to measurerotational motions of large proteins or membrane-bound proteins.Processes on the microsecond or even the millisecond timescale have beenmeasured using phosphorescence, which displays decay times ranging from100 ns to 10 μs. The long lifetimes of specific lanthanide metal-ligandprobes will allow the use of gated detection, which could be employed tosuppress interfering autofluorescence from biological samples and canthus provide further increased sensitivity.

One attractive feature is to combine molecular maps derived from FMTimaging with anatomical tomographic images, e.g., those derived frommagnetic resonance (MR), X-ray computed tomography (CT), ultrasound (US)or even single photon emission tomography (SPECT) or positron emissiontomography (PET) imaging. In particular, the combination with MRI or CTis preferable given the high spatial resolution of these imagingtechniques. DOT imaging (absorption only) has already been combined withMR imaging (Ntziachristos et al., P.N.A.S., USA, 97:2767-72, 1999) whileone of the examples in this application teaches how to combine FMTimaging with MRI. This combination with MRI will enable: (1) thevalidation of FMT imaging in vivo by direct comparison of the MR andoptically acquired images, (2) a direct comparison of cancer appearanceand detection limits based on the anatomical images obtained byT2-weighed MR images, the Gd-enhancement pattern, and molecular activityas resolved with optical imaging, and (3) the implementation of MRstructural and functional information as a priori information in theoptical inversion scheme to obtain highly accurate measures of localizedfluorophore concentration and lifetime. The combination of MRI and FMTalso improves quantitation accuracy of fluorophore concentration andlifetime. Overall, molecular probing will improve the detection accuracyand introduce the ability of molecular target assessment.

To avoid interference with the magnetic field, non-magnetic fiberbundles can be used to transport excitation and emission light to andfrom exciter/detection systems to the patient. For human applications,available commercial or custom-built MRI coils available in any MRfacility can be used. The MR coils can be coupled to one of thegeometries described in FIGS. 3A-3F depending on the application. Toidentify the exact position of the multi-point incident illuminationarray and detector arrays, coupled to the skin, MR or CT imaging itselfcan be used. Knowledge of the spatial location of source fiber ends anddetector fiber ends on uneven surfaces improves optical reconstructions.The skin-coupled fibers as shown in, e.g., FIG. 3E, or internally placedfibers, e.g., endorectally using the array of FIG. 3F, can be detectedby imaging if the arrays are constructed of materials that are uniquelydetectable, e.g., materials that include microreference phantoms filledwith magnetic/x-ray absorbing compounds, certain chemicals, or plastics.For example, to identify the position of the multipoint incidentillumination array and detector array cylinder and the optical fibers onthe MR images, small reference capillaries filled with water and CuSO₄can be attached to the cylinder to appear as bright spots on the MRimages.

FIGS. 4C and 4D are representations of a magnetic resonance (MR) coil 65used for co-registration purposes. The coil 65 is specially built toaccommodate the animal insert 23 shown in FIG. 2B. Two implementationsare considered. In one embodiment, after FMT imaging is performed,insert 23 containing the animal is removed from the imaging chamber 15and positioned within MR coil 65. One or more specially designed glasscapillaries 66 (1 mm glass tubes filled with water and copper sulfate)are attached to insert 23 and enable the MR and FMT images to beco-registered. Such a fiducial marker is visible as a bright circularspot on the left side of the MR image in FIG. 8A (discussed below). FIG.4D shows a mouse positioned within positioning insert 23, within MRIcoil 65. The second embodiment has the coil built directly aroundimaging chamber 15 of FIG. 2A so that concurrent MR and FMT examinationscan be performed.

Data Collection

Five sets of measurements M1-M5 for each of the TR and CW used areobtained as shown in the flowchart of FIG. 5. Although subsets of thecollected data can be used depending on the requirements of theapplication, the highest accuracy is obtained when the full data set isutilized.

As shown in FIG. 5, there are five sets of simple measurements (M1, 71;M2, 72; M3, 73; M4, 74; and M5, 75) to be obtained in initial step 70.In the first step 71, the fluorescence measurement M1, is obtained. Thisis a measurement where the source is scanned at multiple positions, andthe detector acquires the light emitted from the tissue with theband-pass filter on, so that only the emission wavelength (fluorescence)is collected. In step 72, the second measurement, M2, is made as in step71, but without the band-pass filter to acquire the intrinsic signalfrom the tissue at each wavelength. If the fluorescence signal is verysmall compared to the intrinsic signal no filter is required. However,if the fluorescence (M1) from the tissue of investigation is more than1% of M2, then a cut-off filter is used to reject the fluorescencewavelength. In step 73, measurement M3 is made to acquire the amount ofintrinsic light that passes through the fluorescence filter (high-passfilter) used in step 71. To achieve this measurement, the tissue to beinvestigated is removed from the cylinder, and a measurement is madefrom the matching fluid with the fluorescence filter (band-pass filter)on. This measurement is also used to acquire the contribution of ambientlight and other photonic and electronic noise on a per source basis. Instep 74, measurement M4 is obtained with all sources turned off toacquire only the ambient (background) light and CCD noise. In step 75,measurement M5 is obtained without a filter and without tissue at theexcitation and the emission wavelength using appropriate laser diodes.This measurement acquires the background signal.

Practically, for CW measurements each of M1, M2, M3 and M5 vectors is aseries of Ns images (where Ns is the number of sources used). M4 is asingle image of background noise. For the TR measurements, each of M1,M2, M3, and M5, is a set of Ns×Nd×2 wavelengths where Nd is the numberof detectors.

Composite Measurements (CM)

These simple measurements are combined to create self-calibrated (orcomposite) measurements of fluorescence M (step 80 a) and intrinsiccontrast M′ (step 80 b), i.e:

M=(M1−M3)/(M2−M4)  Eq. 1

And

M′=log((M2−M4)/(M5−M4)  Eq. 2

Although not explicit in the above equation, the measurements M1-M5 arefunctions of the frequency. Therefore CW and TR data (after Fouriertransformation) are handled in exactly the same way. The rationale forthis construction is that these composite measurement vectors areindependent of instrumental gain variation, such as differences in theattenuation between different source or detector fibers andinhomogeneities within the CCD chip. Furthermore these vectors subtractfrom the actual measurements systematic errors such as background noise(M4) or high-pass filter imperfectness (M3). Although several ways wouldexist to calibrate the measurements, these particular constructions aredirected after the theoretical predictions of fluorescent and intrinsicsignals, which is a necessary step for quantitative reconstructions.This point is elucidated in the following paragraph.

Depending on the specific application, other alternatives can be used toconstruct self-calibrated composite measurements. For example in dynamicimaging, where the fluorophore concentration and activation is monitoredas a function of time, measurement M5 could be substituted bymeasurement M2 at time 0, preferably before the NIRF probe has beenadministered to the animal. Therefore the fluorochrome absorption can beaccurately monitored as a difference signal from intrinsic tissueabsorption.

Construction of the Forward Problem

To perform tomographic measurements a theoretical prediction of ourcomposite measurements (CM) (i.e., the measurement M and the measurementM′) is required, which is referred to as the “forward predictor” (P) orthe “weight matrix.” The P and CM are combined to produce moleculartomographic measurements as described in the following section. Hereinthe specific theoretical constructions that adapt tomographic principlesof diffraction tomography (Kak & Slaney 1988) are presented.

The P for fluorescence is constructed based on a modified Bornprediction of the forward problem (Li et al., 1996). Generally themedium is assumed to contain a weakly absorbing distribution offluorophores. The fluorophores will be excited by this photondistribution and act as a secondary point source of fluorescent light.The fluorophores as two-level quantum systems and saturation effects areignored because of the small concentration of NIRF probes that areadministered. Then the standard Born expansion for fluorescencemeasurements can be written as:

$\begin{matrix}{{\varphi_{fl}\left( {{\overset{->}{r}}_{d},{\overset{->}{r}}_{s}} \right)} = {\int_{V}{{g_{fl}\left( {\overset{->}{r} - {\overset{->}{r}}_{d}} \right)}\frac{\sigma \; {{cN}_{t}\left( \overset{->}{r} \right)}}{1 - {\; \omega \; \tau}}{\varphi_{0}\left( {\overset{->}{r},{\overset{->}{r}}_{s}} \right)}{\overset{->}{r}}}}} & {{Eq}.\mspace{14mu} 3}\end{matrix}$

where φ_(fl)({right arrow over (r)}_(d),{right arrow over (r)}_(s)) isthe detected fluorescence fluence at position {right arrow over (r)}_(d)for a source at position {right arrow over (r)}_(s), φ₀({right arrowover (r)}−{right arrow over (r)}_(d)) is the established photon fluencein the homogeneous medium due to a source at position, and g_(fl)({rightarrow over (r)}−{right arrow over (r)}_(d)) is a function that describesthe propagation of photons in the diffuse medium at the emissionwavelength. N_(t)({right arrow over (r)})=[F]·γ is the unknownconcentration of the fluorophore F multiplied by the fluorescent yield γat a position {right arrow over (r)}, σ is the absorption cross-sectionof the fluorochrome, c is the speed of light in the diffuse medium,τ=1/Γ is the fluorescent lifetime and ω is the modulation frequency ofthe source light intensity. For sources of constant intensity ω=0. Ourconstruction of the forward predictor (P) in step 88, which predictsmeasurement M (step 80 a) is:

$\begin{matrix}\begin{matrix}{M = \frac{{M\; 1} - {M\; 3}}{{M\; 2} - {M\; 4}}} \\{= {\frac{1}{\varphi_{0}\left( {{\overset{->}{r}}_{d},{\overset{->}{r}}_{s}} \right)}{\int_{V}{{g_{fl}\left( {\overset{->}{r} - {\overset{->}{r}}_{d}} \right)}\frac{\sigma \; {{cN}_{t}\left( \overset{->}{r} \right)}}{1 - {{\omega}\; \tau}}{\varphi_{0}\left( {\overset{->}{r},{\overset{->}{r}}_{s}} \right)}{\overset{->}{r}}}}}}\end{matrix} & {{Eq}.\mspace{14mu} 4}\end{matrix}$

This is a modified Born expansion that normalizes the standard Bornexpansion with the incident field φ₀({right arrow over (r)}_(d),{rightarrow over (r)}_(s)). Therefore, the gain of sources and detectors arecanceled out for each source-detector pair independently.

For intrinsic contrast reconstructions, the forward predictor P′ isdetermined (in step 88) using the Rytov expansion in the frequencydomain, as described, e.g., in O'Leary et al., Opt. Lett. 20:426-428,1995; and Ntziachristos et al., Proc. Natl. Acad. Sci., USA, 97:2767-722000. Then the measurement M′ (step 80 b) can be written as:

$\begin{matrix}\begin{matrix}{M^{\prime} = {\log \frac{{M\; 2} - {M\; 4}}{{M\; 5} - {M\; 4}}}} \\{= {\frac{1}{\varphi_{0}\left( {{\overset{->}{r}}_{d},{\overset{->}{r}}_{s}} \right)}{\int_{V}{{g_{0}\left( {\overset{->}{r} - {\overset{->}{r}}_{d}} \right)}{o\left( \overset{->}{r} \right)}{\varphi_{0}\left( {\overset{->}{r},{\overset{->}{r}}_{s}} \right)}{\overset{->}{r}}}}}}\end{matrix} & {{Eq}.\mspace{14mu} 5}\end{matrix}$

where φ₀ ({right arrow over (r)}, {right arrow over (r)}_(s)) is theincident field from the source at position {right arrow over (r)}_(s) toposition {right arrow over (r)} and o({right arrow over (r)}) is thevector of the unknown absorption and diffusion coefficients changesrelative to the assumed homogeneous background medium.

The functions g_(fl) and φ₀ are calculated by simulating photonpropagation at the emission (step 82) and excitation (step 84)wavelengths for the specific imaging chamber geometry and diffusiontheory. This can be achieved either analytically or numerically. Toperform these simulations, knowledge of the tissue average opticalproperties in the wavelengths of interest are required. The opticalproperties can be obtained by fitting all the intrinsic contrast TRmeasurements to the diffusion model for the appropriate geometry.Analytically, standard methods can be applied (adapted for thecylindrical geometry) as described in Li et al., Appl. Opt.,36:2260-2272 (1997). Here however, we propose to use a homemadefinite-differences numerical algorithm that solves the diffusionapproximation for a cylindrical geometry using a partial boundarycondition (Arridge, Inverse Problems, 15:R41-R93, 1999), whichaccurately models even small source-detector separations. This must beused to obtain more accurate propagation models for the smaller scaleproblem. The only unknown in Eq. 4 and Eq. 5 are then the distributionof the fluorophore or the absorption and diffusion coefficientsrespectively. The minimization of Eqs. 4 and 5 is described in the nextsection.

Data Inversion

Both fluorescence and intrinsic contrast reconstructions are based onthe creation of a function that is subsequently minimized. In step 80 athe composite measurement M is used to construct the functionF(U)=(M−P×U)² and in step 80 b the composite measurement M′ is used toconstruct a function F′(O)=(M′−P′×O)², where U is the vector of unknownnon-quenched (activated) fluorochrome concentration and O is the vectorof unknown absorption and diffusion distributions. The absorptiondistribution can be converted to fluorochrome concentration via theBeer-Lambert Law. The matrices P, P′ are described in the previoussection. In steps 92 a and 92 b, the functions F(U) and F′(O) areminimized to obtain the distribution and magnitude of U and O,respectively. The minimization is obtained using algebraicreconstruction techniques although any other minimization method can meused to find the solution of the constructed functions.

Iteration is not necessary when only small amounts of fluorochrome areactivated. This is the most typical case. However, if for certainapplications large concentrations of activated fluoro chrome areexpected (namely the absorption perturbation yielding more than 10%variation in the intrinsic signal), then iterative steps are necessary.The first step of the iterative process 95 assumes a homogeneousbackground with the average optical properties of the medium ofinvestigation. Subsequent steps use images U and O as background maps inthe creation of matrices P, P′. When iteration is used, the creation ofP, P′ using numerical solutions of the diffusion equation is necessary.Iteration is also necessary when the background distribution of thefluorochrome is comparable to the contrast obtained from localized areasof high accumulation such as the tumor. Iteration is typically stoppedwhen each iteration step does not significantly change the calculatedresult.

Molecular Maps

The new systems and methods enable the quantitative, three-dimensionalcalculation of molecular and molecular-activation maps. The resolvedimage U contains the concentration of fluorescing or activatedfluorochrome, whereas the absorption image contained in O is aquantitative representation of the total fluorochrome concentration(quenched and de-quenched). The ratio of activated over totalfluorophore concentration is the activation ratio map (step 93):

AR=U/O  Eq. 6

which represents the amount of activation normalized by the amount offluorochrome actually distributed in the volume of investigation. Forvolumes in which the absorption is zero the ratio AR is not defined.This is natural, since for zero fluorochrome distribution there shouldbe no activation. Therefore, the ratio AR is by default applied only inthe volume elements with non-zero absorption.

The generation of a molecular map (reporting the activity of the enzymetrypsin) is shown in FIGS. 6A-6C. A molecular map is a representation ofan endogenous process or molecule. A molecular map (MM) is bestdescribed as MM=k*AR, where k is a constant; i.e., MM=k*(U/O).

FIG. 6A is an image of an absorption map, showing the concentration of amolecular probe sensitive to degradation by trypsin. The bright spot inFIG. 6A is a representation of the total amount of the probe, both thequenched and the unquenched fractions. FIG. 6B is the correspondingfluorescence map, which measures only the fraction of de-quenched (i.e.,enzyme activated) trypsin sensitive probe. FIG. 6C provides the ARimage, or “molecular map,” displaying the “fluorescence activation” asan image where the bright spot is directly proportional to the amount ofadded trypsin enzyme used in this experiment.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 FMT Images of a Phantom

In one embodiment, phantom experiments were performed to verify thethree-dimensional position and accuracy of measurements. Theexperimental set-up is illustrated in top view in FIG. 7A. Briefly, aphantom 100 containing 3 capillary tubes (1 mm internal diameter) 103,was constructed using a triangular geometry and inserted into theoptical chamber (15 in FIG. 2A) containing a turbid medium 102 (0.5%Intralipid® in water). The capillaries 103 were separated 8 and 11 mmfrom each other as shown in FIG. 7A, and were coated with a blackfluorochrome to maximize absorption. The capillaries were imaged threedimensionally.

FIG. 7B depicts the reconstructed image 110 at a plane perpendicular tothe longitudinal axis of the imaging chamber 15, at about the middle ofthe three-dimensional volume imaged. The high contrast allowed forhigh-resolution imaging of the three capillaries with high positionalprecision. The reconstruction mesh used was 0.8×0.8×2 mm³. Thereconstruction used 24 sources×36 detectors.

Example 2 FMT Images of Trypsin Activity Over Time

In another experiment, quantitative, spatially localized information onfluorescence activation was obtained as a function of time. As shown inFIGS. 8A and 8B, a 3 mm tube 123 was immersed in a tissue-like fluid(Intralipid®) to form phantom 102. The tube contained 1.5 μM of a Cy5.5probe, which was activated by the addition of the enzyme trypsin intothe tube at time 0. Only a single plane was imaged in this experiment bysequentially illuminating each of twelve light emitting points in thedirection of curved arrow 125. FIG. 8B illustrates the phantom in athree-quarter view.

FIGS. 8C to 8F illustrate a series of axially reconstructed framesobtained at different time points. The frames show the probe activationas a function of time. For example, as shown in FIG. 8C, at 20 minutesafter trypsin was added to the capillary, only ˜20% of the probe hadbeen activated. However, as shown in FIG. 8F, at 200 minutes after theenzyme was added, ˜75% of the probe had been activated. Each frame wasacquired by sequentially directing light in each one of twelve sourcefibers located on the same plane along the cylinder. For each source,the CCD acquired light from the detector fibers for 5 seconds. The totalacquisition time per frame was therefore 1 minute (12 sources×5 secondseach).

Example 3 Multiple Co-Registered Images of Cathepsin B Activity in aMouse

In another experiment, combined MR/FMT imaging was used to obtain mapsof cathepsin B protease activity in human tumors implanted in nude mice(FIGS. 9A-(C). The tumors were cathepsin B rich HT1080 fibrosarcoma,which had been implanted into the mammary fat pad 7-10 days prior to theexperiment. The animals received an IV injection of a cathepsin Bsensitive imaging probe (Weissleder et al., Nat. Biotechnol.,17:375-378, 1999) at 24 hours prior to the imaging experiments. Theanimals were anaesthetized with an intraperitoneal injection of 90 mg/kgketamine and 9 mg/kg xylazine and were placed into the insert 23 shownin FIG. 2B. The insert and animal were placed into the imaging chamber15 and measurements M1, M2, M3, and M4 were obtained.

Subsequently, the mouse within the insert was removed from the imagingchamber. Fiducials (as described herein) with water were attached topredetermined positions on the periphery of the insert. The insert wassubsequently placed in the MR coil and a set of axial T2-weighted imagedwere obtained. The role of the fiducials was to identify on the MRimages the position of selected source and detector fibers for laterco-registration of the images. The fiducial (a glass capillary tubearranged longitudinally along the outside cylinder wall) shown on theslices of FIGS. 9A, 10A, and 11A as a bright spot on the left side ofthe image, for example, indicates the position of detectors 1, 13, and25 on the corresponding slices.

The results show an MR image (FIG. 9A), a cathepsin B molecular map(FMT)(FIG. 9B), and one of the MR slices fused with the FMT image toproduce a combined MR/molecular map (FIG. 9C). There is excellentcongruence of optical and MR contrast from the images obtained at thetumor level. The tumor demonstrates strong molecular activity ofcathepsin B (fluorescence activation), corroborated byimmunohistochemistry and Western blotting. The co-registration of thefluorescent activation and T2 image are shown on the fused image in FIG.9C. The remaining two rows of images are slices that show cathepsin Babsence and/or presence in other tissues. Specifically, FIGS. 10A and10B show an MR image and FMT image at heart level, respectively. Asexpected, there is no cathepsin B activity in the lung and heart, andthus nothing lights up on the FMT image in FIG. 10B. FIGS. 11A and Bshow an MR image and FMT image at kidney level, respectively. Thefluorochrome appearing in the kidney is likely excreted excess, and doesnot reflect cathepsin activity.

This is an example where a subset of the full measurement array is used(only M1 through M4 CW measurements, no M5 and no TR data) to produce aenzymatic activity image that conveys useful information for theclinical examples describes in the following examples. Moreover, thisseries of images in FIGS. 9A to 11B confirms that the new methods andsystems can be used to generate multi-slice images of living animals.

Example 4 Molecular Maps

To demonstrate the use of producing molecular maps we have used theset-up of Example 3, but acquired the full array of M1 through M5measurements in CW mode. The optical properties of Intralipid® wereindependently measured with a time-resolved system. Then a fluorescencemap (FIG. 6B) collected 50 minutes after trypsin activation and anabsorption map (FIG. 6A) were constructed according to the algorithmdescribed in the flowchart in FIG. 5. The molecular map (AR image)calculated according to step 93 is shown in FIG. 6C and demonstrates 40%activation of the enzyme sensitive probe 50 minutes after activation.

Example 5 Enzyme-Specific Probes

We have synthesized a number of different sensitive enzyme-specificimaging probes useful for FMT imaging. The probes are specific forcathepsin D, cathepsin K, the enzymatically active form of prostatespecific antigen (PSA), and matrix metalloprotease-2, among otherenzymes. The specificity of these probes was shown by incubation withpurified or recombinant human enzymes and by measurement of fluorescenceactivation in a fluorometer. The NIR fluorophore Cy5.5 was used as aquenched reporter in all of these probes. Any of these probes can beused in animals and human patients as described herein to measure enzymeactivity within deep tissues (both normal and diseased tissues). Forexample, MMP-2 activity can be measured in tumors before and aftertreatment with an MMP-2 inhibitor (e.g., Prinomastat®). Suchmeasurements of molecular target assessment are useful for rapid drugefficacy screening in vivo in animal models. Moreover, such screeningmethods can be used to assess the efficacy of a particular therapy in aspecific patient.

Example 6 Clinical Use

The new FMT methods are expected to have broad clinical implications.One use is for early detection of disease at a stage when molecularabnormalities are present, but have not yet led to phenotypicabnormalities (e.g., mutations in cancers which have not yet produced atumor mass). Another use is for molecular target assessment in diseasedtissues (1) to determine if a given target is present in a patient(e.g., level of expression of a protease), (2) to determine whether anexperimental drug has an effect on its intended molecular target invivo, (3) to individualize and tailor treatments for a given patient,and (4) to optimize the dose of a given molecular drug for a givenpatient. In this sense, the new FMT imaging methods are an adjunct totesting drug efficacy. Such measurements would also be of value in aclinical setting to determine the effects of enzyme inhibitor drugs,receptor blockers, and other molecular drugs. The methods could be usedto monitor a wide variety of disease including cancer, cardiovasculardisease, AIDS, infection, immunologic diseases, inflammation,dermatological and ophthalmic diseases, neurodegenerative disease andothers.

Example 7 Multiple Probes

The new FMT methods can be performed with the concomitant use ofmultiple molecular probes (each with their own, specific excitation andemission wavelengths) to report multiple molecular abnormalities duringthe same FMT imaging acquisition. The described system can be adapted byadding one or more new laser sources to excite the additionalfluorescent molecular probes. Imaging signals are collected throughappropriate filter systems, making sure that there is not spectraloverlap among the different channels. Image reconstruction, algorithms,and displays are similar to those for single wavelength imagingdescribed herein.

Example 8 Frequency Domain Technology

The TR system described herein can be modified by using one or morefrequency domain sources, preferably at multiple frequencies. Thetheoretical formulation is written in the frequency domain so that theuse of frequency technology is directly applied to the existingalgorithms. The rationale behind using frequency domain technology issimilarly to TR technology in that it yields multi-frequency informationthat can differentiate absorption and scattering in intrinsic contrastmode and fluorophore concentration and life-time in fluorescence mode.If frequency technology is used, the instrument in FIG. 4A issubstituted by sources modulated at one or several frequencies anddetection channels that are responsible for signal demodulation, such aslock-in amplifiers or preferably quadrature demodulators, similar to theones used for the detection of MR signals.

Example 9 Differential Dynamic Imaging (DDI)

The implementation of the composite measurements described above can beapplied in several ways to obtain fluorescent and intrinsic contrast,and to construct the AR images. For example, whereas a general scheme ofan animal injected with a NIRF molecular probe is considered in Eqs. 4and 5, one could obtain measurements from an animal before injection ofthe NIRF probe and then obtain differential measurements of absorptionand fluorescent contrast after NIRF probe injection. This technique hasimportant applications in monitoring the kinetics of uptake andactivation (as also demonstrated in Example 2). This approach alsoyields the most accurate results since differential measurements allowfor the reconstruction of the fluorochrome/chromophore absorptionindependently of background absorption (since only the absorption changecan be reconstructed). Therefore, more accurate AR maps can be producedas a function of time.

Example 10 Imaging at Multiple Wavelengths

An alternative implementation of composite measurements than the oneused in Example 9 is to employ four or more wavelengths for eachmeasurement set. For N tissue chromophores, N or more of thesewavelengths are selected at a spectral region where the NIRF probe doesnot absorb. Therefore, true “intrinsic” contrast is obtained, i.e.,contrast that is due only to the natural tissue chromophoreconcentrations. Using the spectral information of these chromophores,one can calculate their absorption at the emission and excitationwavelengths of the NIRF probe. The other two wavelengths are used toconstruct absorption images at the excitation and emission wavelengths.Those latter images reconstruct absorption due to both the naturaltissue chromophore concentration and the fluorochrome distribution. Bysubtracting the images obtained at the excitation or emission wavelengthfrom the absorption images calculated only for the tissue naturalchromophores, one can obtain the true fluorochrome/chromophoreconcentration.

Example 11 Clinical FMT System

The new systems and methods described herein are easily applied to aclinical setting. For example breast cancer detection can be achievedwith a circular/cylindrical multipoint incident illumination array orwith a compression/planar array. Brain measurements can be made with anelastic band of optical fibers attached to the scull or aplanar/reflectance geometry could be applied. See FIGS. 3A-F for variousarrays. The described FMT imaging methods can be conducted sequentiallyor simultaneously with MR or CT measurements, because the opticaltechnology is compatible with other radiological modalities.

In a clinical setting, CW measurements would be useful for theeconomical collection of large numbers of measurements. However, even alimited number of more advanced technologies (e.g., IM or TR asdescribed above) can significantly improve the information content ofthe CW measurements. However, it is envisaged that a clinical system canbe built entirely based on CW technology. As frequency-domain ortime-domain technologies become cheaper, the whole system can be basedonly on frequency-domain or time-domain technologies.

Other Embodiments

A subcategory of the general reconstruction scheme of molecularactivation described herein would be the use of simple transilluminationof tissue for the detection of molecular events. This is a relaxation ofthe tomographic imaging to simple projection imaging, similar, but notsame as the one described previously for reflectance imaging (Weisslederet al., U.S. Pat. No. 6,083,486). Transillumination allows formeasurements of absorbers of fluorochromes through the whole tissue,therefore it achieves penetration of several centimeters, in contrast toreflectance imaging that can penetrate only for a few centimeters at themost. Transillumination of molecular events cannot three-dimensionallyresolve or quantify molecular activity but can still be used to monitorqualitatively relative changes of molecular activation.

In another embodiment, the new systems and methods can be used to imageendogenous fluorescence in an animal. For example, a gene encoding afluorescent protein, such as green fluorescent protein or fluorescein,can be included adjacent to a gene of interest that is to be expressedin an animal or human patient using standard gene therapy techniques.The expression of the gene of interest can be determined indirectly byimaging the fluorescent protein. If this protein is expressed, then thegene of interest has also been expressed.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-29. (canceled)
 30. A method of obtaining a three-dimensional,quantitative tomographic image of a target region within a patient oranimal body, the method comprising: administering a near-infraredfluorescent probe to the patient or animal body, wherein the probeselectively accumulates within a target region in the patient or animalbody; directing near-infrared excitation light into the patient oranimal body at multiple locations, thereby transilluminating the patientor animal body; detecting at multiple points excitation lighttransmitted through the patient or animal body; detecting fluorescentlight emitted from the patient or animal body; and processing thedetected excitation light and the detected fluorescent light to providea three-dimensional tomographic image that corresponds to thethree-dimensional target region within the patient or animal body and tothe quantity of the probe accumulated in the target region.
 31. Themethod of claim 30, wherein the near-infrared light directed into thepatient or animal body is at a wavelength of from 550 nm to 950 nm. 32.The method of claim 30, wherein the near-infrared light directed intothe patient or animal body is at a wavelength of from 670 nm to 850 nm.33. The method of claim 30, wherein the excitation light is continuouswave light.
 34. The method of claim 30, wherein processing the detectedexcitation light and the detected fluorescent light to provide thethree-dimensional tomographic image comprises iteratively solving forfluorescent probe distribution using a model of excitation lightpropagation and emission light propagation through the patient or animalbody.
 35. The method of claim 30, wherein the fluorescent probecomprises a fluorochrome attached to a delivery vehicle, wherein thedelivery vehicle comprises any one or more of a polymer, a dendrimer, aprotein, a carbohydrate, a lipid sphere, and a nanoparticle.
 36. Themethod of claim 30, wherein the probe comprises a member selected fromthe group consisting of an activatable fluorescent probe, a targetedfluorescent probe, a receptor-targeted near-infrared fluorochrome, anantibody-targeted near-infrared fluorochrome, a wavelength-shiftingbeacon, a multi-color fluorescent probe, and a lanthanide metal-ligandprobe.
 37. The method of claim 30, wherein the probe has at least one ofthe following properties: the probe becomes activated upon targetinteraction; the probe becomes deactivated after target interaction; theprobe changes its quantum yield upon target interaction; the probechanges its fluorescence lifetime after target interaction; the probechanges its fluorescent spectrum after target interaction; and the probehas high binding affinity to a target in the target region.
 38. Themethod of claim 30, comprising the step of disposing the patient oranimal body within an imaging chamber prior to directing thenear-infrared excitation light into the patient or animal body.
 39. Themethod of claim 30, further comprising generating a surfacerepresentation of the patient or animal body, wherein the surfacerepresentation comprises an identification of positions of incidentillumination and positions of detection, and using the surfacerepresentation in processing the detected excitation light and thedetected fluorescent light to provide the three-dimensional tomographicimage.
 40. A fluorescence-mediated tomographic imaging systemcomprising: a near-infrared excitation light source; an imaging chamberconfigured to direct the near-infrared excitation light into a patientor animal body disposed within the chamber at multiple locations,thereby transilluminating the patient or animal body; a detectorconfigured to detect at multiple locations excitation light transmittedthrough the patient or animal body and fluorescent light emitted from aprobe within the patient or animal body; and a processor configured toprocess data corresponding to the detected excitation light transmittedthrough the patient or animal body and the detected fluorescent lightemitted from the probe within the patient or animal body to provide athree-dimensional tomographic representation of a region within thepatient or animal body and of the quantity of the probe accumulated inthe target region.
 41. The system of claim 40, wherein the near-infraredexcitation light source produces light at a wavelength of from 550 nm to950 nm.
 42. The system of claim 40, wherein the near-infrared excitationlight source produces light at a wavelength of from 670 nm to 850 nm.43. The system of claim 40, wherein the near-infrared excitation lightsource is configured to produce continuous wave light.
 44. The system ofclaim 40, wherein the near-infrared excitation light is a continuouswave laser.
 45. The system of claim 40, wherein the processor isconfigured to process the data corresponding to the detected excitationlight and the detected fluorescent light using a forward model of (i) anexcitation field from the near-infrared excitation light source to theprobe within the patient or animal body and (ii) an emission field fromthe probe within the patient or animal body to the detector.